• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of neoplasiaLink to Publisher's site
Neoplasia. Sep 2002; 4(5): 380–387.
PMCID: PMC1661677

Functional Inactivation of pRB Results in Aneuploid Mammalian Cells After Release From a Mitotic Block1


The widespread chromosome instability observed in tumors and in early stage carcinomas suggests that aneuploidy could be a prerequisite for cellular transformation and tumor initiation. Defects in tumor suppressors and genes that are part of mitotic checkpoints are likely candidates for the aneuploid phenotype. By using flow cytometric, cytogenetic, and immunocytochemistry techniques we investigated whether pRB deficiency could drive perpetual aneuploidy in normal human and mouse fibroblasts after mitotic checkpoint challenge by microtubule-destabilizing drugs. Both mouse and human pRB-deficient primary fibroblasts resulted, upon release from a mitotic block, in proliferating aneuploid cells possessing supernumerary centrosomes. Aneuploid pRB-deficient cells show an elevated variation in chromosome numbers among cells of the same clone. In addition, these cells acquired the capability to grow in an anchorage-independent way at the same extent as tumor cells did suggesting aneuploidy as an initial mutational step in cell transformation. Normal Mouse Embryonic Fibroblasts (MEFs) harboring LoxP sites flanking exon 19 of the Rb gene arrested in G2/M with duplicated centrosomes after colcemid treatment. However, these cells escaped the arrest and became aneuploid upon pRB ablation by CRE recombinase, suggesting pRB as a major component of a checkpoint that controls cellular ploidy.

Keywords: pRB, cell cycle control, CIN, centrosomes, aneuploidy


Cancer cells are characterized not only by mutations in specific oncogenes and tumor-suppressor genes, but they also display gain and loss of chromosomes as well as alterations in chromosome number (aneuploidy) [1–4]. Chromosomal instability (CIN) [2] could account for the heterogeneity observed in individual tumors and could underlie the clonal differences seen in the same solid tumor. Recently, it has been suggested that CIN in cancer cells could be associated with mutations in mitotic checkpoint genes [5]. Near diploid tumor cells without the hSecurin gene, a component of the complex of proteins responsible for chromatid cohesion, became aneuploid over several generations generations [6]. However, to date inactivation of hSecurin has not been observed in human cancers suggesting that altered expression of other genes must be responsible for the aneuploidy observed in cancer. Also, it has been shown that mammalian cells with reduced expression of MAD2 show CIN even though additional mutations are likely necessary for cellular transformation to occur readily [7,8].

An alternative view is that CIN is not generated by gene mutation/s; instead it could result from an initial abnormal chromosome complement within the cell [9]. This hypothesis comes from studies that showed that treatments with nongenotoxic drugs induce a near-complete aneuploidy [10].

Previously we showed that normal human fibroblasts lacking functional pRB re-replicate their DNA when challenged with antimicrotubule drugs [11]. These cells probably do not go through a “point of no return”; instead they decondense their chromosomes and return to a G2 phase [12,13] from which they can re-replicate their DNA. Here we show that transient polyploidization, by uncoupling DNA replication and cytokinesis, can trigger CIN both in human and murine pRB-deficient primary fibroblasts and that cells derived following colcemid treatment are capable of unanchored growth in soft agar to the same extent as tumor cells. By using mouse embryonic fibroblasts (MEFs) harboring LoxP sites flanking exon 19 of the Rb gene [14], we demonstrate a direct relationship between pRB ablation, centrosome duplication, and generation of aneuploid cells.

Materials and Methods

Cell Culture

Normal human fibroblasts IMR90 (diploid human embryonic lung fibroblasts, ATCC CCL-186), stably expressing a neomycin resistance gene (IMR90neo), or neomycin resistance and the gene encoding the human papillomavirus type 16 (HPV16) E7 protein (IMR90E7) were obtained by retroviral gene transduction. Cells expressing E7 have nonfunctional RB protein. MEFs with conditional Rb alleles (RbLoxP/LoxP MEFs) were kindly provided by Dr. Marco Crescenzi (Istituto Superiore di Sanità, Rome). RbLoxP/LoxP MEFs were infected (MOI: 300, which ensures 90% to 95% of successful infected fibroblasts) with the empty vector J-pCA13 or with adenoviruses (AdCre) expressing the Cre recombinase under control of the human cytomegalovirus (hCMV) immediate-early promoter to generate MEFs devoid of pRB (Rb-/- MEFs). All adenoviruses used in this study were replication-deficient (E1 region deleted). Cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Euroclone, Milan, Italy).

Cell Cycle Analysis

Asynchronously growing cells were treated with 0.2 µg/ml colcemid (Demecolcine, Sigma) for 72 hours and then released into complete medium without colcemid. DNA content was determined using PI staining alone by treating the cells with PBS containing 4 µg/ml of PI and 40 µg/ml RNase. BrdU incorporation (10 µM for 4 hours) was used to determine DNA synthesis of the cells released in medium without colcemid. Analysis of BrdU-labeled cells was conducted as described previously [15] and samples were analyzed on a Becton Dickinson (NJ, USA) FACScan. Experiments were repeated twice; 5000 events were analyzed for each sample, and representative experiments are shown.

Fluorescence In Situ Hybridization

Centromeric probes specific for chromosome 2 and 6 (kindly provided by Dr. Mariano Rocchi, University of Bari, Italy) were used for interphase and metaphase fluorescence in situ hybridization (FISH). FISH was done following published procedures [15] with minor modifications. Briefly, after RNase and proteinase K treatment and DNA denaturation in 70% formamide/2x SSC, cells were dehydrated in an ethanol series. Following overnight incubation at 37°C in a hybridization mixture consisting of 50% deionized formamide, 5% dextrane sulfate, 2x SSC, 1% Tween 20, 100 µg/ml salmon sperm DNA, and 100 ng biotinylated and digoxigenated pX2 (chr. 2) and pEDZ6 (chr. 6) probes, the slides were washed and blocked in 4x SSC/0.5% nonfat dry milk. Slides were layered with FITC-conjugated avidin or antidigoxigenin rhodamine (Roche Molecular, Mannheim, Germany) in 4x SSC/0.5% blocking reagent. Slides were stained with Hoechst 33258 (0.05 µg/ml), examined on a Zeiss (Gottingen, Germany) Axioskop microscope equipped for fluorescence, and the observed nuclei and metaphases were captured on a CCD camera (Zeiss). Images were transferred to Adobe PhotoShop for printing.

Determination of Ploidy

Asynchronous cells were treated with 0.2 µg/ml colcemid (Demecolcine, Sigma) for 4 hours. Cells were harvested by trypsinization, swollen in 75 mM KCl at 37°C, fixed with 3:1 methanol:acetic acid (v:v), and dropped onto clean, ice-cold glass microscope slides. The slides were air dried and stained with 3% Giemsa in phosphate-buffered saline for 10 minutes. Chromosome numbers were evaluated using a Zeiss Axioskop microscope under a 100x objective.

Immunofluorescence Microscopy

To visualize multipolar spindles, cells grown on rounded glass coverslips were treated with Taxol (Paclitaxel, Sigma) 1 µg/ml for 2 hours and then fixed with 3.7% formaldehyde for 10 minutes at 37°C, permeabilized with 0.1% Triton X (Sigma) in PBS for 15 minutes, and blocked with 0.1% BSA for 30 minutes, both at room temperature. Coverslips were incubated with a mouse monoclonal antibody against β-tubulin (Sigma; diluted 1:200 in PBS) overnight at 4°C, followed by a goat anti-mouse IgG-FITC conjugated secondary antibody (Sigma; diluted 1:100 in PBS) for 1 hour at 37°C. For immunostaining of centrosomes of asynchronously grown cells, we used a mouse monoclonal antibody against γ-tubulin (Sigma; diluted 1:4000 in PBS) incubated overnight at 4°C. Cells were washed in PBS buffer and exposed to a FITC-conjugated goat anti-mouse IgG secondary antibody (Sigma; diluted 1:100 in PBS) for 1 hour at 37°C. Nuclei were visualized with Hoechst 33258 (0.05 µg/ml) and examined on a Zeiss Axioskop microscope equipped for fluorescence and images were captured with a CCD digital camera (Zeiss). Images were transferred to Adobe PhotoShop for printing.

Immunoblot Analyses

For immunoblot analyses, MEFs were lysed in SDS/PAGE sample buffer, protein extracts were resuspended in loading buffer (0.125 M Tris-HCl, 4% SDS, 20% v/v glycerol, 0.2 M dithiothreitol, 0.02% bromphenol blue, pH 6.8), and 60 µg of protein (as determined by the Bradford assay) was loaded per lane on a 6% SDS PAGE gel. After gel electrophoresis proteins were electrotransferred onto a Hybond C membrane (Amersham Life Science, Milan, Italy). Electroblotted gel was stained with Coomassie blue and the membrane was reversibly stained with Ponceau S to verify equal loading and transfer. The membrane was blocked in 5% (w/v) nonfat milk in TBST buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween 20) at room temperature and incubated overnight at 4°C with the anti-pRB primary antibody (C-15, Santa Cruz Biotechnology, Santa Cruz, CA) 1 µg/ml in blocking solution. After three washes with TBST buffer, the blot was incubated in horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology, diluted 1:5000) for 1 hour at room temperature and washed again three times with TBST buffer. Blot was developed with chemiluminescent reagents (SuperSignalWest Pico, Pierce Rockford, IL) and exposed to CL-Xposure film (Pierce, Rockford, IL) for 1 to 5 minutes.

Focus Assay

We have used a base layer (2 ml) composed of DMEM, 20% FBS, and 1% Noble agar. Noble agar was allowed to sit in six-well multidish plates of 35-mm diameter (Iwaki, Bibby Sterlin Staffordshire, England, UK). This substrate was overlaid with 2 ml of a second layer of agar media (0.3% DMEM, 20% FBS) containing a suspension of 10,000 cells/dish and incubated at 37°C in a 5% CO2 atmosphere. Foci were visible within 10 to 14 days.


pRB-Deficient Human Fibroblasts Become Aneuploid upon Release from Colcemid and Show Potential for Transformation

Previously, we showed that pRB deficiency in human and mouse fibroblasts leads to increased ploidy in the presence of spindle inhibitors due to re-replication of most, if not all, of the genome without inducing DNA damage [11]. This initial abnormal chromosome complement could be the basis for generating CIN in human cells when associated with malfunction of a checkpoint controlling the correct ploidy or reduplicated centrosomes. To further examine this hypothesis, both normal (IMR90neo) and pRB-deficient (IMR90E7) human fibroblasts were treated with the antimicrotubule drug colcemid for 72 hours to induce transient polyploidization. The DNA content and distribution in E7-expressing cells showed, by FACScan analyses, that the majority of the cells accumulated with a 4 N (70%) and 8 N DNA content (12%) (Figure 1A). In contrast, there were no IMR90neo cells with an 8 N DNA content, and 36% of cells accumulated with a 4 N DNA content. Bivariate FACScan analyses (BrdU vs propidium iodide [PI] incorporation) of IMR90E7 cells colcemid treated for 72 hours and then released for 4 hours in complete medium without colcemid, showed that additional 12% of IMR90E7 cells (Figure 1B) were able to enter S phase with respect to those analyzed at 72 hours. The cell cycle profiles showed that these additional cells in S-phase (BrdU positive) originated from the cells with a 4 N DNA content that re-replicated their DNA (data not shown). On the contrary, IMR90neo cells treated and released in the same way did not show any BrdU incorporation at the indicated times.

Figure 1
Effects of mitotic arrest on DNA replication and ploidy in IMR90neo and IMR90E7 human cells. (A) FACS profiles of PI-stained IMR90neo and IMR90E7 cells untreated or treated with colcemid (0.2 µg/ml). (B) Histograms summarizing cell cycle analyses ...

We next asked whether these treated cells (henceforth designated IMR90neo-t and IMR90E7-t) were capable of further growth when released into complete medium without the drug. During the first week after the release, we estimated that about 40% of IMR90E7-t cells died. However, in the second week of culture the remaining cells proliferated at the same rate as IMR90E7 cells (parental) when cultured in the absence of the mitotic inhibitor. In contrast, IMR90neo-t cells proliferated very slowly, becoming completely arrested within 2 weeks following the release. We did chromosome counts on 100 metaphase spreads of these IMR90E7-t cells to ascertain the ploidy status. Cytogenetic analysis showed more than 90% of the cells were aneuploid (Figure 1D) and contained different numbers of chromosomes per metaphase (range of chromosomes per metaphase 47 to 80). In contrast, all IMR90E7 and IMR90neo cells examined were diploid. Fluorescence in situ hybridization (FISH) with chromosome-specific anti-centromeric probes, both on metaphases and nuclei, confirmed differences in chromosome numbers between untreated and treated E7-expressing cells (Figure 1C). We isolated several single-cell clones from the population of colcemid-released IMR90E7-t cells from two independent experiments, to determine ploidy in pRB-deficient cells in which DNA replication is uncoupled from cytokinesis. Clones were allowed to expand, and after 1 week of growth FACScan analyses showed that the DNA content for all of them was greater than that of the parental cells, indicating that these cells had acquired a perpetual aneuploidy (Figure 2A). Alteration of ploidy in these cells was confirmed by determining chromosome numbers (Figure 2B). Cytogenetic analyses of pRB-deficient cells released from the colcemid block and analyses of single-cell clones showed high variability in chromosome numbers from cell to cell suggesting random occurrence of chromosomal gain and loss. These observations were consistently confirmed during at least 3 months of cell culture. The observed heterogeneity in chromosome numbers in these pRB-deficient cells suggests that the initial trigger (transient polyploidy) results in chromosomal instability that allows aneuploidy cells to proliferate and perpetuate.

Figure 2
Cell cycle analyses and features of IMR90E7-t derived clones. (A) FACS profiles of asynchronous PI-stained IMR90E7 and IMR90E7-t derived clones showing the presence of hyperdiploidy in all the clones isolated. (B) Chromosome/metaphase numbers in IMR90E7-t ...

Aneuploidy in tumors has been associated with the presence of multipolar mitotic spindles strictly correlated with alterations in centrosome numbers. We examined the spindle organization in these growing aneuploid cells by β-tubulin staining (Figure 2C) and found multipolar spindles in all of the cells scored. Chromosomal instability (CIN) is a hallmark of cancer and accounts for the heterogeneity observed in tumor cells. However, the possibility that the acquired aneuploidy is an initial event in the transformation process is still debated. Therefore, we characterized the aneuploid IMR90E7-t population and the independently derived aneuploid cell clones for their potential for transformation. Both the IMR90E7-t population and cell clones grew in soft agar as discrete colonies indicating the capability of these cells to grow in an anchorage-independent manner. Estimation of the number of foci (Figure 2D) revealed that these cells have a potential for transformation comparable to that displayed from the tumor cells DLD1 used as a positive control. In contrast, wild-type IMR90neo and IMR90E7 cells failed to grow in soft agar (Figure 2D).

pRB Ablation Allows Growth of MEFs Arrested with Duplicated Centrosomes

It has been shown that centrosome duplication requires both E2F and CDK2/ cyclin A in Chinese hamster ovary cells [16], and that stable expression of HPV16-E7 oncoprotein in keratinocytes, but not in fibroblasts [17], causes induction of numerical centrosome abnormalities that could represent an early event during neoplastic progression potentially driving genomic destabilization [18]. However, HPV16-E7 oncoprotein causes inactivation of other cellular targets such as p107 and p130 pRB's family protein, p21WAF1-Cip1 [19] and insulin-like growth factor binding protein 3 (IGFBP-3) [20]. Then, we determined whether the lack of pRB only is sufficient for allowing perpetual aneuploidy associated with centrosome reduplication in mammalian cells.

To this aim we used MEFs carrying conditional Rb alleles (RbLoxP/LoxP MEFs) in which two LoxP sites are inserted into the introns surrounding exon 19 of the Rb gene [14]. We infected RbLoxP/LoxP MEFs with adenoviruses expressing the Cre recombinase (AdCre) to excise the exon 19 and to generate cells with truncated pRB (Rb-/- MEFs), which are functionally equivalent to Rb null cells. RbLoxP/LoxP MEFs were also infected with adenoviruses that did not express the Cre transgene to generate isogenic murine cells (pRB proficient, indicated as RbLoxP/LoxP) to be used as a control. Western blot experiments (Figure 3A) showed that as early as 96 hours RbLoxP/LoxP MEFs infected with AdCre were completely devoid of pRB when compared to the same cells infected with adenoviruses that did not express the Cre transgene. Infected cells were allowed to expand and 2 weeks after they were colcemid treated. We determined by FACScan analysis the DNA content of both RbLoxP/LoxP and Rb-/- MEFs after exposure to colcemid. Figure 3B shows typical flow cytometry results at 0 and 72 hours. Colcemid-treated (time 72 hours) RbLoxP/LoxP MEFs showed increase in 4 N cells but not in 8 N cells when compared to untreated (time 0 hours) RbLoxP/LoxP MEFs that showed the majority of the cells with a 2 N DNA content. As expected PI analyses revealed that untreated Rb-/- MEFs were predominantly 2 N or 4 N with very few >4 N cells. In contrast the majority of Rb-/- MEFs treated with colcemid accumulated with a 4 N and >4 N of DNA content. Additional cytogenetic analyses showed that more than 50% of these colcemid-treated Rb-/- MEFs were polyploid (Figure 3C). Evaluation of centrosome numbers, by γ-tubulin detection, in these colcemid-treated Rb-/- MEFs revealed that 42% of cells had more than two centrosomes, 46% had two centrosomes, and only 12% had one centrosome. This indicates that DNA re-replication, because of lack of pRB, is accompanied by centrosome reduplication.

Figure 3
Effect of mitotic arrest on DNA replication and ploidy in RbloxP/loxP and Rb-/- MEFs. (A) Western blot analysis showing the level of pRB expression after 96 hours from the adenoviral infection: (lane 1): pRbloxP/loxP MEF infected with the empty vector ...

Then, we examined the fate of the treated cells by releasing both RbLoxP/LoxP and Rb-/- MEFs (henceforth indicated as RbLoxP/LoxP MEFs-t and Rb-/- MEFs-t) from the colcemid block. We observed that RbLoxP/LoxP MEFs-t remained arrested for at least 2 weeks. In contrast, Rb-/- MEFs-t proliferated as did IMR90E7-t cells (Figure 3E). Cytogenetic analyses performed 4 weeks after the release from the block imposed by colcemid showed that the great majority of the Rb-/- MEFs-t were aneuploid (75%) suggesting that lack of pRB allowed aneuploid cells to proliferate. The staining of these cells for β-tubulin revealed the presence of high numbers of tripolar and multipolar spindles (Figure 3D). To examine whether RbLoxP/LoxP MEFs-t cells, which experienced a prolonged if not permanent arrest upon colcemid treatment, were able to escape the arrest following pRB ablation, they were infected with the adenovirus AdCre to excise by recombination exon 19 of the Rb gene and thus inactivate it. These cells (indicated as RbLoxP/LoxP MEFs-t+AdCre) escaped the arrest and started to proliferate similarly to R-/- MEFs-t (Figure 3E). Cytogenetic analyses revealed that more than 50% of these cells become aneuploid. In addition, centrosome analysis (Figure 3F, Table 1), at 1 week after the infection, by γ-tubulin staining of RbLoxP/LoxP MEFs-t+AdCre cells showed an increase in cells harboring two centrosomes (55%) and more than two centrosomes (20%, range 3 to 8), and only 25% of the cells with one centrosome. On the contrary, RbLoxP/LoxP MEFs-t cells showed that 58% and 37% of the cells had one or two centrosomes, respectively, as expected whether treated cells are arrested in G1 and G2/M, and only 5% of RbLoxP/LoxP MEFs-t cells had more than two centrosomes. Both untreated RbLoxP/LoxP and Rb-/- MEFs showed a similar percentage of cells with one or two centrosomes. The increasing number of cells harboring supernumerary centrosomes in RbLoxP/LoxP MEFs-t+AdCre suggests that pRB ablation allow cells arrested with at least two centrosomes and with altered ploidy to escape the arrest.

Table 1
Centrosome Analysis in Mouse Embryonic Fibroblasts.


A current paradigm in cancer research states that cancer originates from a single founder cell in which mutations in few crucial genes have occurred. However, this paradigm does not seem to be in accordance with either epidemiological data or with multiple mutations and chromosomal aberrations, both structural and numerical, which are found in the majority of tumors. These chromosomal alterations are believed to destabilize the genome and further increase the occurrence of new changes [9]. Aneuploidy observed in the majority of tumors and in early stage carcinomas strongly suggests that it could be considered a prerequisite in initiation as well as in tumor progression [2,3]. The occurrence of transient polyploidization caused by DNA rereplication [11] could be considered a trigger for generating CIN and perpetual aneuploidy in human cells [10]. Our findings both in human and murine cells suggest that pRB could be a major component of a pathway that controls ploidy when the mitotic checkpoint is activated by antimicrotubule drugs. Our results, showing that pRB-deficient cells are able to tolerate aneuploidy and in turn acquire the ability to grow in an anchorage-independent way, support the hypothesis that aneuploidy may be a driving force in tumorigenesis rather than a side effect of the transformation process driven by mutations in oncogenes and tumor-suppressor genes [21]. Although changes in chromosome numbers might be caused by alterations in specific genes of the mitotic checkpoint [6,8,22], our results suggest that also pRB is playing an important role in this process. In fact, when the mitotic checkpoint is challenged, pRB deficiency predisposes cells to aneuploidy by DNA re-replication [11], and the concomitant centrosome reduplication is necessary to maintain and perpetuate the resulting aneuploid cells. In addition, these findings suggest that lack of pRB is necessary to allow aneuploid cells to proliferate despite the presence of supernumerary centrosomes, which are thought to cause mitotic failure, producing nonviable daughter cells [23]. Mammalian cells that do not have mitotic checkpoints defects, when exposed to spindle inhibitors for a long time, eventually adapt and exit mitosis, escaping the inhibitor-induced arrest. Recently, it has been shown that p53 competent cells exposed to antimicrotubule drugs, after adaptation, arrest in a G1-like cell cycle phase [24]. Surprisingly, p53-deficient human fibroblasts (expressing the HPV-16 E6-oncoprotein) that showed polyploidy after 72 hours of colcemid exposure, arrested with the subsequent passages in culture when released in medium without colcemid (L.L. and A.D.L., unpublished observations). These findings suggest the existence of a pathway dependent on pRB, likely activated in normal diploid fibroblast adapted cells, which is able to monitor alterations in cellular ploidy or centrosome duplication, thereby inducing a prolonged cell cycle arrest.

Many reports have suggested the existence of a relationship between reduplicated centrosomes and the presence of gross aneuploidy. However, it is still debated whether centrosome alterations are a cause of genomic instability or a consequence of alterations in the cell cycle of cancer cells [25]. The ability of both HPV-16 E7 oncoprotein and E2F together with CDK2/cyclin A to induce centrosome duplication in mammalian cells [16,17] suggests that abrogation of pRB functions may have similar effects on centrosome homeostasis that could drive subsequent aneuploidy in tumor cells. Our findings that in proliferating human fibroblasts lack of pRB, caused by HPV16-E7 oncoprotein expression, did not affect centrosome number differently from that previously observed in keratinocytes [17] likely reflects differences in cell types used. This suggests that pRB dysfunction does not have any apparent deleterious effect on centrosomes in asynchronously growing fibroblasts. However, our results that RbLoxP/LoxP MEFs-t arrested for 2 weeks with a 4 N DNA content and duplicated centrosomes were able to escape the arrest only after pRB ablation, suggest the existence of a checkpoint, in which pRB plays a central role, which monitors centrosome numbers and when activated arrests cells in a G1-like cell cycle phase.

Finally, our results support the following scenario in mammalian cells. Duplicated centrosomes reduplicate in mitotic arrested cells whether a new round of DNA synthesis occurs because of pRB dysfunction. When the mitotic block is over, the checkpoint is activated by the presence of supernumerary centrosomes and the signal transmitted to pRB, which halts further cell cycle progression. On the contrary pRB-deficient cells could progress in the cell cycle and undergo cytokinesis with more than one centrosome inherited by each daughter cell. Then, some of the aneuploid pRB-deficient cells are capable to proliferate and perpetuate both the CIN phenotype and centrosome hypertrophy.


We are grateful to Marco Crescenzi (Istituto Superiore di Sanità, Rome) for providing us with MEFs harboring conditional Rb alleles and advice on adenoviral infection. Floxed MEFs were derived from mice generated in A. Berns' Laboratory (Netherland s Cancer Institute, Amsterdam).


mouse embryonic fibroblasts
human papillomavirus type 16
propidium iodide
fluorescence in situ hybridization


This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), Ministero dell'Istruzione dell'Università\ della Ricerca (MIUR), and University of Palermo (ex 60%).


1. Sen S. Aneuploidy and cancer. Curr Opin Oncol. 2000;12:82–88. [PubMed]
2. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998;396:643–649. [PubMed]
3. Shih IM, Zhou W, Goodman SN, Lengauer C, Kinzler KW, Vogelstein B. Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis. Cancer Res. 2001;61:818–822. [PubMed]
4. Li R, Sonik A, Stindl R, Rasnick D, Duesberg P. Aneuploidy vs. gene mutation hypothesis of cancer: recent study claims mutation but is found to support aneuploidy. Proc Natl Acad Sci USA. 2000;97:3236–3241. [PMC free article] [PubMed]
5. Cahill DP, da Costa LT, Carson-Walter EB, Kinzler KW, Vogelstein B, Lengauer C. Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics. 1999;58:181–187. [PubMed]
6. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, Peters JM, Kinzler KW, Vogelstein B, Lengauer C. Securin is required for chromosomal stability in human cells. Cell. 2001;105:445–457. [PubMed]
7. Wang X, Jin DY, Wong YC, Cheung AL, Chun AC, Lo AK, Liu Y, Tsao SW. Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis. 2000;21:2293–2297. [PubMed]
8. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W, Dobles M, Sorger PK, Murty VV, Benezra R. MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells. Nature. 2001;409:355–359. [PubMed]
9. Duesberg P, Rasnick D, Li R, Winters L, Rausch C, Hehlmann R. How aneuploidy may cause cancer and genetic instability. Anticancer Res. 1999;19:4887–4906. [PubMed]
10. Duesberg P, Rausch C, Rasnick D, Hehlmann R. Genetic instability of cancer cells is proportional to their degree of aneuploidy. Proc Natl Acad Sci USA. 1998;95:13692–13697. [PMC free article] [PubMed]
11. Di Leonardo A, Khan SH, Linke SP, Greco V, Seidita G, Wahl GM. DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function. Cancer Res. 1997;57:1013–1019. [PubMed]
12. Rieder CL, Cole RW. Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase. J Cell Biol. 1998;142:1013–1022. [PMC free article] [PubMed]
13. Scolnick DM, Halazonetis TD. Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature. 2000;406:430–435. [PubMed]
14. Marino S, Vooijs M, van Der Gulden H, Jonkers J, Berns A. Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev. 2000;14:994–1004. [PMC free article] [PubMed]
15. Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J. Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA. 1988;85:9138–9142. [PMC free article] [PubMed]
16. Meraldi P, Lukas J, Fry AM, Bartek J, Nigg EA. Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A. Nat Cell Biol. 1999;1:88–93. [PubMed]
17. Duensing S, Lee LY, Duensing A, Basile J, Piboonniyom S, Gonzalez S, Crum CP, Munger K. The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc Natl Acad Sci USA. 2000;97:10002–10007. [PMC free article] [PubMed]
18. Duensing S, Duensing A, Crum CP, Munger K. Human papillomavirus type 16 E7 oncoprotein-induced abnormal centrosome synthesis is an early event in the evolving malignant phenotype. Cancer Res. 2001;61:2356–2360. [PubMed]
19. Jones DL, Alani RM, Munger K. The human papillomavirus E7 oncoprotein can uncouple cellular differentiation and proliferation in human keratinocytes by abrogating p21Cip1-mediated inhibition of cdk2. Genes Dev. 1997;11:2101–2111. [PMC free article] [PubMed]
20. Mannhardt B, Weinzimer SA, Wagner M, Fiedler M, Cohen P, Jansen-Durr P, Zwerschke W. Human papillomavirus type 16 E7 oncoprotein binds and inactivates growth-inhibitory insulin-like growth factor binding protein 3. Mol Cell Biol. 2000;20:6483–6495. [PMC free article] [PubMed]
21. Duesberg P, Rasnick D. Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskeleton. 2000;47:81–107. [PubMed]
22. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature. 1998;392:300–303. (see comments) [PubMed]
23. Brinkley BR. Managing the centrosome numbers game: from chaos to stability in cancer cell division. Trends Cell Biol. 2001;11:18–21. [PubMed]
24. Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic checkpoint following spindle disruption. Mol Cell Biol. 1998;18:1055–1064. [PMC free article] [PubMed]
25. Stearns T. Centrosome duplication. A centriolar pas de deux. Cell. 2001;105:417–420. [PubMed]

Articles from Neoplasia (New York, N.Y.) are provided here courtesy of Neoplasia Press
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...